Continued Development of Techniques for In Vivo Investigation of the Function of Identified Neurons One of our major aims is to understand how circuitry within the basal ganglia contributes to the control and learning of actions. An important component of this line of research is gaining an understanding of the activity of specific neuronal subtypes within this circuitry during the learning, planning, initiation and performance of actions and action sequences. We have worked with Drs. Rui Costa of the Champalimaud Neuroscience Institute and Steven Vogel of the Laboratory of Molecular Physiology at NIAAA to develop a technique for in vivo fiber photometry to perform Time Correlated Single Photon Counting (TCSPC) fluorimetry in the striatum in vivo. We are now extending this technique to measure calcium transients in presynaptic elements in awake, freely-moving mice using a new single-fiber chronically-implantable photometry system that we recently developed. To this end, we have expressed the genetically-encoded calcium indicator GCaMP6 in two different cortical regions, the primary motor cortex (M1) and the medial prefrontal cortex (mPFC). This results in GCaMP6 expression in the axon projections from M1 neurons to the dorsolateral or sensorimotor striatum (DLS), and from mPFC neurons to the dorsomedial or associative striatum (DMS). We verified with the TCSPC system that fluorescence can be detected in the appropriate striatal subregions. We then trained animals to perform a skill learning task, the rotarod task which is an elevated rotating dowel on which the mouse must learn to walk. We have measured fluorescent signals that indicate increased presynaptic calcium, likely indicative of increased activity and neurotransmitter release, in both the M1-DLS and mPFC-DMS pathways during early sessions of rotatod training. The M1-DLS activity appears to increase with the velocity of rod rotation/movement. With extensive training over several days, the mPFC-DMS activity is almost completely abolished despite greatly improved skill performance. The M1-DLS activity also decreases with training/skill improvement, but is still quite prominent in the latter stages of training. These findings indicate that both associative and sensorimotor corticostriatal circuits are engaged during early stages of skill learning, but the mPFC component of the associative circuit may no longer be involved once the skill is well learned. The M1-DLS activity changes indicate possible honing/consolidation of activity in a major component of the sensorimotor system during well-learned skill performance. These findings fit well with our earlier studies showing progressively greater control by the sensorimotor circuit with training in this task, and are consistent with findings from others indicating consolidation of M1 neuronal firing with increasing mastery of skills. These findings have implications for action control and treatment of movement disorders. In future experiments we hope to modify activity in these different pathways and determine the effects at different stages of skill learning. We will also work to identify molecular mechanisms underlying the changes in cortical input. This approach can also be applied to measurement of presynaptic function in a variety of in vivo settings. D2 Receptor Roles in Synaptic Activity and Striatal-based Behaviors Along with the Alvarez laboratory here at NIAAA, we are examining the roles of different striatal dopamine receptors in synaptic function, learning and memory, and response to abused substances. Given the prominent roles of dopamine in action control, motivation, reward, reinforcement and addiction, understanding the role of dopamine receptors is important to gain a better grasp of a variety of neural functions and neural disorders. Dopamine D2 receptors are expressed on a variety of cells and cellular elements in the striatum. One prominent site of expression is the striatal medium spiny projection neurons (MSNs) that give rise to the striatopallidal pathway. These constitute approximately half of all striatal MSNs. We have used a Cre-loxP based strategy to remove D2 receptors from these MSNs. These mice show impaired rotarod performance and learning. They also appear to be deficient in some associative learning tasks. To determine how this MSN-D2 deletion affects synaptic events that might contribute to learning, we examined endocannabinoid-dependent long-term depression (LTD) at corticostriatal synapses in the DLS region in brain slices. This form of synaptic plasticity is known to require D2 receptors, but the locus of the important pool of receptors is not known. Preliminary data indicate that LTD is intact in mice lacking MSN-D2Rs, and that this plasticity is still blocked by D2 and CB1 cannabinoid receptor antagonists. Thus, the D2 receptors important for corticostriatal LTD may not reside on MSNs. We have also deleted the receptors from cholinergic striatal interneurons (CINS) using a similar strategy. These mice show strong impairment in the ability to learn an instrumental lever-pressing task, and we are currently attempting to determine what task components contribute to this poor learning. In ongoing experiments, we are also examining LTD in brain slices from the CIN D2R-deleted mice. Determining the locus of D2Rs important for LTD and striatal-based behaviors will help us to determine how LTD might contribute to action learning, and should aid in more targeted pharmacological and neurobiological treatments for the variety of disorders involving the striatum.